Device for tendon and ligament treatment

ABSTRACT

Devices and methods for treating defects in connective tissue are provided along with methods for making such devices. The devices can include acellular arterial tissue matrices that facilitate regrowth of the damaged tissue.

This application claims priority to U.S. Provisional Application No.61/558,328, filed Nov. 10, 2011, which is incorporated herein byreference in its entirety.

The present disclosure provides improved devices and methods fortreating defects in connective tissue, such as tendons and ligaments.

The healing of tendons and ligaments is often associated with severescarring. Furthermore, during the healing process, these various formsof connective tissue can sometimes adhere to other surrounding fibroustissues. Scar formation and adhesion can limit the range of motion ofthe healed tissue and alter its material properties. Due to scarformation and/or adhesion within the joint, greater loading forces maybe required to achieve desirable motion.

A conduit that protects the damaged tendon or ligament from furtherinjury may expedite the healing process as well as minimize adhesion orscar formation. The conduit may aid the healing process by creating aprotective environment and by minimizing the loss of growth factors orother natural cytokines that are released during healing.

In certain embodiments, a device for treating connective tissue isprovided. The device comprises an arterial tissue matrix, whereinsubstantially all the native cells have been removed, and wherein thearterial tissue matrix is derived from an arterial section that has beencut across its length, and wherein the arterial tissue matrix includesan intact basement membrane present in the arterial section from whichthe tissue matrix is derived.

In certain embodiments, a method for preparing an arterial tissue matrixdevice is provided. The method comprises selecting an arterial section,removing substantially all the native cells from the section to obtainan arterial tissue matrix, without removing a luminal basement membranefrom the arterial section, and cutting the arterial section across itslength.

According to certain embodiments, a method for treating a tendon orligament is provided. The method comprises selecting a tendon orligament in need of treatment, selecting an arterial tissue matrix fromwhich substantially all the native cells have been removed, andcontacting the arterial tissue matrix with at least a portion of thetendon or ligament in need of treatment.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method of treating a tendon or ligament, accordingto certain embodiments.

FIG. 2 illustrates a device for treating tendons or ligaments, accordingto various embodiments.

FIG. 3 illustrates a device for treating tendons or ligaments, accordingto various embodiments.

FIG. 4 is a flow chart summarizing the various steps that may be used toproduce a device for performing methods of the present disclosure,according to certain embodiments of the disclosed method.

DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Reference will be made in detail to certain exemplary embodimentsaccording to the present disclosure, certain examples of which areillustrated in the accompanying drawings. Wherever possible, the samereference numbers will be used throughout the drawings to refer to thesame or like parts.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. In addition, terms such as “element” or “component”encompass both elements and components comprising one unit and elementsand components that comprise more than one subunit, unless specificallystated otherwise. Also, the use of the term “portion” may include partof a moiety or the entire moiety. Any range described herein will beunderstood to include the endpoints and all values between theendpoints.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

As used herein, the term “acellular tissue matrix” refers generally toany tissue matrix that is substantially free of native cells. Acellulartissue matrices can be derived from human or xenogenic sources.Acellular tissue matrices may be seeded with exogenous cells derivedfrom the recipient or other sources.

FIG. 1 illustrates a method of treating a tendon or ligament, accordingto certain embodiments. According to certain embodiments, the methodcomprises selecting a tendon 104 or ligament in need of treatment, andcontacting an arterial tissue matrix 106 with at least a portion of thetendon or ligament in need of treatment 105. As described further below,the arterial tissue matrix can include an acellular arterial tissuematrix from which substantially all the native cells have been removed.

Tendons and ligaments are particular types of connective tissue that arecomposed primarily of fibrous collagen. While tendons connect muscle tobone, ligaments connect bone to bone, as found within a joint. Tendonsserve to transmit force generated by the muscles, while ligaments serveto stabilize the associated joint.

In various embodiments, the tissue matrices of the present disclosurecan be used to treat a defect in any tendon or ligament. For example,the matrices can be used to treat tendons or ligaments associated withany joint, including the joints of the fingers, wrists, elbows,shoulder, knees, ankles, hips, feet, or any other joint in the body. Insome embodiments, the tendon or ligament can be an elongate tubulartendon or ligament, which can be subject to significant tensilestresses. For example, the tendon or ligament can include any tendon,including a rotator cuff tendon, an anterior cruciate ligament, and aflexor or extensor tendon of the hand, wrist, ankle, or foot.

Various defects occurring in tendons and ligaments can be treated withthe disclosed methods. A defect may result from various sources,including, but not limited to, an acute or chronic injury, a geneticpre-condition, age, inadequate nutrition, or body weight. These variousfactors may contribute to defective tissue by either applying excessiveforce to the tendon or ligament or by serving to weaken their structuralproperties in some manner. Ligaments are subject to injury whenstretched, strained, or stressed beyond their normal range of motion.Defects to tendons and ligaments may include, but are not limited tostrains, tears, and gaps. Injuries particular to tendons includetendinitis, i.e., inflammation of the tendon, and tendinosis, in whichmicrotears pervade the connective tissue. In FIG. 1, a defect comprisinga tear 105 in a wrist tendon 104 is shown.

The arterial tissue matrix can contact the tendon or ligament in variousways in order to effect treatment. In some embodiments, contacting thetissue comprises positioning the tissue matrix across the area in needof treatment, i.e, the area comprising the defect. In other embodiments,contacting the tissue comprises wrapping the arterial tissue matrixaround the area in need of treatment.

The tissue matrices disclosed herein can possess certain attributes thatare desirable in the treatment of damaged tendons or ligaments. Thedisclosed tissue matrix contains elastin, which is found in arterialtissue, as well as collagen. Due to the combination of elastin andcollagen within the tissue matrices, the disclosed device is elastic,and able to resume its original shape after being stretched orcompressed. The elastic nature of the device also makes it highlyresistant to tearing, yet very flexible. The collagen/elastinarchitecture also confers load-bearing properties to the device.

The disclosed tissue matrix can be used in various ways related to thetreatment of tendons or ligaments. The device can provide a protectivebarrier or conduit around the tendon or ligament, which serves toprotect the damaged area from further injury. In some embodiments, thebarrier can also prevent scar formation as well as prevent adhesion ofoutside tissue with the damaged tendon or ligament. When used as aprotective barrier, the device can also help prevent fibrosis, in whichan excessive amount of fibrous tissue results during the healingprocess. The barrier may also serve to minimize the loss of growthfactors and cytokines released during the healing process, therebyfacilitating regeneration of damaged tissue.

Once the tissue matrix has contacted the area of tendon or ligament inneed of treatment, various methods can be used to fix the placement ofthe device. In some embodiments, the tissue matrix is sutured in place.In other embodiments, glue or clips can be used to fix the position ofthe device. Additional means of fixing the device derived from thesurgical or medical arts can also be used in accordance with the claimedmethod so long as they do not impede the formation of a protectivebarrier.

In various embodiments, the device disclosed herein can also include asurface comprising an intact basement membrane, a typical component ofarterial vessels. The basement membrane is a thin fibrous sheet,composed of basal and reticular lamina, which serves to support theepithelium and endothelium found within the body. Epithelium lines thecavities and surfaces of organs, while endothelium lines the interiorsurface of blood vessels. In normal arteries, an intact basementmembrane lines the interior surface of the artery, also known as theluminal surface.

The presence of an intact basement membrane provides further mechanicalsupport and also provides a smooth surface on one side of the device.When the basement membrane is positioned on the outer surface of thedevice, its smooth surface prevents adhesion from occurring. This may bedesirable when damage to the tendon or ligament is severe and healingrequires a longer period of time. In other instances, it may bedesirable to position the basement membrane on the interior surface ofthe device. The positioning of the basement membrane at the interior orexterior surface of the device can be determined as needed.

In various embodiments, an arterial tissue matrix is cut along itslength. Prior to cutting, the arterial section has a cylindrical ortubular shape, as it is found in the body. Upon cutting along itslength, the arterial section is able to unfurl into a relatively flat,two-dimensional surface, i.e, a sheet. The sheet can be of any shape,such as a circle, square, rectangle, etc., and can be further cut asneeded to facilitate certain medical procedures. For example, FIG. 2illustrates a device 201 for treating tendons or ligaments, according tovarious embodiments.

In certain embodiments, shapes can be formed with the arterial tissuematrix using various methods. As described further below, partialdehydration can be used to render the tissue matrix more pliable, a moldcan be used to provide the desired shape, and irradiation can be used tofix the new shape.

In certain embodiments, a bioactive substance is added to the arterialtissue matrix which further facilitates the repair, regrowth, orregeneration of tendon or ligament. The bioactive substance can be addedto the tissue matrix at any point prior to implantation of the tissuematrix in a subject. For example, a bioactive substance can be addedduring preparation of the device but prior to its storage. In otherembodiments, a bioactive substance may be added to the device afterstorage, but immediately prior to its implantation into a subject.Bioactive substances include, but are not limited to antimicrobialagents, cytokines, growth factors, anti-inflammatory agents, steroids,and corticosteroids. In some embodiments, bioactive substances caninclude various types of tissue, for example, adipose tissue.

In some embodiments, the bioactive substance comprises cells thatfacilitate the repair, regrowth, or regeneration of tendon or ligament.In certain embodiments, the acellular tissue matrices can be seeded withstem cells, such as mesenchymal stem cells, including embryonic stemcells and adult stem cells harvested from bone marrow, adipose tissue,and neuronal cells. In other embodiments, autologous stem cells may beused. In some embodiments, allogenic cells can be pre-seeded to thegrafts and cultured in vitro and lysed before implantation.

In various embodiments, the tissue matrix can be produced from anarterial section from a variety of different anatomic sites, including,but not limited to, carotid, femoral, ulnar, median, and/or brachialarteries. Furthermore, arterial tissue can be derived from human and/ornon-human sources, such as pigs, as described in more detail below. Incertain embodiments, the section of artery is selected based on adesired size (e.g., length of defect to be treated and/or approximatetubular diameter of connective tissue to be treated). In variousembodiments, the length of the defect can be greater than 1 cm, greaterthan 2 cm, between 0.1 cm and 1 cm, between 1 cm and 2 cm, greater than4 cm, greater than 6 cm, greater than 10 cm, or any ranges in between.

As shown in FIG. 2, the device 201 can be shaped to facilitate placementand/or to improve visualization during surgery. For example, as shown,the device 201 can include one or more indentations 202 formed along thecorners of the tissue matrix. The indentations can allow improvedvisualization of the underlying tissue in need of treatment, forexample, a tendon 104 with a tear 105. In addition, when the device iswrapped around the damaged tissue with the side comprising theindentations facing up, the surgeon can easily see the extent to whichone end of the device overlaps the other, facilitating suturing.

In certain embodiments, the devices of the present disclosure can beproduced with a predefined shape or structure to conform to the anatomicsite to be treated. For example, FIG. 3 illustrates a device 301 fortreating tendons or ligaments, according to various embodiments. Asshown, the device 301 includes a tubular shape, that can be preselectedto conform to the shape and/or dimensions of selected ligaments ortendons. In addition, the device 301 can include other features, such asa basement membrane, bioactive substance, or any other properties orstructures discussed herein. As discussed previously, the basementmembrane can be positioned at the interior surface 107 or exteriorsurface 108 of the device.

In certain embodiments, a method of preparing a device for treatingconnective tissue is provided. The method comprises selecting anarterial section, removing substantially all the native cells from thesection to obtain an arterial tissue matrix without removing a luminalbasement membrane from the arterial section, and cutting the arterialsection along its length.

While an acellular tissue matrix may be made from the same species asthe acellular tissue matrix graft recipient, different species can alsoserve as tissue sources. Thus, for example, an acellular tissue matrixmay be made from porcine tissue and implanted in a human patient.Species that can serve as recipients of acellular tissue matrix anddonors of tissues or organs for the production of the acellular tissuematrix include, without limitation, mammals, such as humans, nonhumanprimates (e.g., monkeys, baboons, or chimpanzees), pigs, cows, horses,goats, sheep, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats,or mice.

Arterial cellular tissue matrices suitable for use in the presentdisclosure can be produced by a variety of methods. In variousembodiments, the arterial acellular tissue matrices can include variousproteins other than collagen, which may support tendon or ligamentregeneration. In some embodiments, the matrices can includeglycosaminoglycans (GAGs) and/or elastins, which are present in intactarterial tissue. In other embodiments, the matrices can include anintact basement membrane.

Generally, the steps involved in the production of an acellular tissuematrix include harvesting the tissue from a donor (e.g., a human cadaveror animal source) and cell removal under conditions that preservebiological and structural function. In certain embodiments, the processincludes chemical treatment to stabilize the tissue and avoidbiochemical and structural degradation together with or before cellremoval. In various embodiments, the stabilizing solution arrests andprevents osmotic, hypoxic, autolytic, and proteolytic degradation,protects against microbial contamination, and reduces mechanical damagethat can occur with tissues that contain, for example, smooth musclecomponents (e.g., blood vessels). The stabilizing solution may containan appropriate buffer, one or more antioxidants, one or more oncoticagents, one or more antibiotics, one or more protease inhibitors, andone or more smooth muscle relaxants.

The tissue is then placed in a decellularization solution to removeviable cells (e.g., epithelial cells, endothelial cells, smooth musclecells, and fibroblasts) from the structural matrix without damaging thebiological and structural integrity of the collagen matrix. Thedecellularization solution may contain an appropriate buffer, salt, anantibiotic, one or more detergents (e.g., TRITON X100™, sodiumdeoxycholate, polyoxyethylene (20) sorbitan mono-oleate), one or moreagents to prevent cross-linking, one or more protease inhibitors, and/orone or more enzymes. In some embodiments, the decellularization solutioncomprises 0.1% to 10% (w/v) TRITON-X-100™. In other embodiments, thedecellularization solution comprises 0.5% to 5% (w/v) TRITON-X-100™. Insome embodiments, the decellularization solution comprises 1% TRITONX-100™ in RPMI media with Gentamycin and 25 mM EDTA(ethylenediaminetetraacetic acid). In some embodiments, the tissue isincubated in the decellularization solution overnight at 37° C. withgentle shaking at 90 rpm. In certain embodiments, additional detergentsmay be used to remove fat from the tissue sample. Additional detergentsmay comprise sodium deoxycholate in some embodiments. In certainembodiments, the decellularization solution comprises 0.1% to 10% sodiumdeoxycholate. In other embodiments, 2% sodium deoxycholate is added tothe decellularization solution.

It would be understood that variations can be made in the protocol aboveand still be within the scope of the present invention. For example,other physiological buffers can be utilized so long as they do notimpede the decellularization process. In some embodiments, suitabledetergents can include, but are not limited to, SDS, sodium cholate,sodium deoxycholate, TRITON-X-100™, and NP40™. The selected detergentscan be used at a range of concentrations, for example, from 0.1 to 10%(w/v), 0.5 to 2.0%, 1.0 to 2.0%, 0.1 to 2%, 0.5 to 5%, 0.5 to 10%, 0.1to 2%, or any values within those ranges.

After the decellularization process, the tissue sample is washedthoroughly with saline. In some exemplary embodiments, e.g., whenxenogenic material is used, the decellularized tissue is then treatedovernight at room temperature with a deoxyribonuclease (DNase) solution.In some embodiments, the tissue sample is treated with a DNase solutionprepared in DNase buffer (20 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 20 mM CaCl₂ and 20mM MgCl₂. In some embodiments, the DNase solution comprises 1 to 100units/ml DNase. In certain embodiments, the solution comprises 30units/ml DNase. Optionally, an antibiotic solution (e.g., Gentamicin)may be added to the DNase solution. In some embodiments, the solutioncomprises 1 to 200 μg/ml gentamicin. In other embodiments, the solutioncomprises 50 μg/ml gentamicin. Any suitable buffer can be used as longas the buffer provides suitable DNAse activity.

Elimination of the α-gal epitopes from the collagen-containing materialmay diminish the immune response against the collagen containingmaterial. The α-gal epitope is expressed in non-primate mammals and inNew World monkeys (monkeys of South America) as well as onmacromolecules such as proteoglycans of the extracellular components. U.Galili et al., J. Biol. Chem. 263:17755 (1988). This epitope is absentin Old World primates (monkeys of Asia and Africa and apes) and humans,however. Id. Anti-gal antibodies are produced in humans and primates asa result of an immune response to α-gal epitope carbohydrate structureson gastrointestinal bacteria. U. Galili et al., Infect. Immun. 56:1730(1988); R. M. Hamadeh et al., J. Clin. Invest. 89:1223 (1992).

Because non-primate animals (e.g., pigs) produce α-gal epitopes,xenotransplantation of collagen-containing material from these mammalsinto primates often results in rejection because of primate anti-galbinding to these epitopes on the collagen-containing material. Thebinding results in the destruction of the collagen-containing materialby complement fixation and by antibody dependent cell cytotoxicity. U.Galili et al., Immunology Today 14:480 (1993); M. Sandrin et al., Proc.Natl. Acad. Sci. USA 90:11391 (1993); H. Good et al., Transplant. Proc.24:559 (1992); B. H. Collins et al., J. Immunol. 154:5500 (1995).Furthermore, xenotransplantation results in major activation of theimmune system to produce increased amounts of high affinity anti-galantibodies. Accordingly, in some embodiments, when animals that produceα-gal epitopes are used as a tissue source, the substantial eliminationof α-gal epitopes from cells and from extracellular components of thecollagen-containing material, and prevention of cellular α-gal epitopere-expression can diminish the immune response against thecollagen-containing material associated with anti-gal antibody bindingto α-gal epitopes.

To remove α-gal epitopes, after washing the tissue thoroughly withsaline to remove the DNase solution, the tissue sample may be subjectedto one or more enzymatic treatments to remove certain immunogenicantigens, if present in the sample. In some embodiments, the tissuesample may be treated with an α-galactosidase enzyme to eliminate α-galepitopes if present in the tissue. In some embodiments, the enzymatictreatment may comprise 50 to 500 U/L α-galactosidase prepared in asuitable buffer. In some embodiments, the tissue sample is treated withα-galactosidase at a concentration of 200 or 300 U/L prepared in 100 mMphosphate buffer at pH 6.0. In other embodiments, the concentration ofα-galactosidase is increased to 400 U/L for adequate removal of theα-gal epitopes from the harvested tissue. Any suitable enzymeconcentration and buffer can be used provided adequate removal ofantigens is achieved.

Alternatively, rather than treating the tissue with enzymes, animalsthat have been genetically modified to lack one or more antigenicepitopes may be selected as the tissue source. For example, animals(e.g., pigs) that have been genetically engineered to lack the terminalα-galactose moiety can be selected as the tissue source. Fordescriptions of appropriate animals, see co-pending U.S. applicationSer. No. 10/896,594 and U.S. Pat. No. 6,166,288, the disclosures ofwhich are incorporated herein by reference in their entirety.

As an optional step, the matrix may then be subjected to partialdehydration to facilitate shaping the tissue into a desired form afterthe tissue matrix has been decellularized and treated to remove certainantigenic epitopes. Partial dehydration of tissues has been found torender the collagen fibers of the tissue matrix more pliable, thusfacilitating their reorientation. Excessive means of dehydration such asfreeze-drying can render tissues too brittle for further processing andis therefore, undesirable.

A variety of suitable processes can be used to partially dehydrate thearterial tissue matrix. Examples of suitable methods include blot-dryingwith water-absorbent towels, isothermal water desorption with controlledhumidity, application of mechanical forces to tissues, centrifugation,and/or controlled and/or directional freezing. Any method can be used topartially dehydrate the matrix as long as the method does not causeundesirable tissue alterations.

After partial dehydration, mechanical forces can be applied to thearterial matrix to reorient the collagen fibers within the matrix. Thiscan be done in a variety of ways, so long as forces are directed in sucha way to produce a final desired shape. In some embodiments, a moldhaving a desired shape is produced, and a sheet of arterial tissuematrix is placed in contact with the mold. Forces are then applied toportions of the sheet to reorient fibers to a shape consistent with themold. In certain embodiments, to produce a stable tubular product suchas a conduit, a dowel rod or other tubular structure is selected as amold, and a sheet of arterial tissue matrix is wrapped around the mold.

After the collagen fibers are reoriented, the arterial tissue matrix maybe treated to stabilize the new structure. In some embodiments, thestructure is stabilized by exposing the matrix to radiation. Radiationmay cause a small degree of cross-linking sufficient to produce a stablethree-dimensional structure. Excessive cross-linking may alter thebiological properties of tissue products and is therefore, undesirable.After irradiation, the stable structure will tend to conform to a shapesimilar to the mold (or shape of the matrix at the time of irradiation),but will be sufficiently flexible to allow the tissue product to bemanipulated during surgery. Suitable forms of radiation include gammaradiation, e-beam radiation, and X-ray radiation.

In various embodiments, the disclosed methods can further compriseadditional processing of the device. Such processing of the device mayinclude disinfecting or sterilization the arterial tissue matrix. Insome embodiments, the arterial tissue matrix is disinfected withisopropyl alcohol (IPA) (e.g., at about 70% IPA).

The disclosed devices can be further treated to produce aseptic orsterile materials. Accordingly, in various embodiments, the devices canbe sterilized after preparation. As used herein, a “sterilizationprocess” can include any process that reduces bioburden in a sample, butneed not render the sample absolutely sterile.

Certain exemplary processes include, but are not limited to, a gammairradiation process, an e-beam irradiation process, ethylene oxidetreatment, and propylene oxide treatment. Suitable sterilizationprocesses include, but are not limited to, those described in, forexample, U.S. Patent Publication No. 200610073592A1, to Sun et al.; U.S.Pat. No. 5,460,962 to Kemp; U.S. Patent Publication No. 2008/0171092A1,to Cook et al. In some embodiments, sterilization is performed inconjunction with packaging of the device, while in other embodiments,sterilization can occur after packaging. In certain embodiments,sterilization is combined with the fixing step.

After the devices are prepared by the disclosed methods, they may bestored for some time before implantation in or on a patient. In certainembodiments, the device may be packaged in a TYVEK® pouch for storagepurposes. The device may also be stored in different states, forexample, in a wet state or in a freeze-dried state.

In some embodiments, the device is freeze-dried after preparation.Terminal sterilization by ionizing radiation conducted afterfreeze-drying, however, can potentially damage freeze-dried tissue. Tominimize this damage, the processed material can be packaged wet inmoisture-permeable TYVEK® bags first. The tissue is then irradiated in awet state. After irradiation, the wet samples can be freeze-dried forstorage.

A sample protocol in accordance with the disclosed methods is providedin FIG. 4. The order of certain steps can be changed as needed. Certainsteps may also be discarded as necessary. Specific details regardingeach step are provided throughout the present disclosure. A section ofan arterial vessel is procured from a suitable source 401. The arterialsection is still an intact tube at this stage. The arterial section isthen cut across its length and unfurled into a sheet 402. The sheet isthen incubated in an appropriate solution to decellularize the tissue403. The sheet is then treated with enzymes to remove DNA and α-galepitopes 404. After enzyme treatment, the sheet is further re-sized asdesired 405. The sheet is then subjected to partial dehydration whichfacilitates molding of the sheet by rendering it more pliable 406. Thesheet is then placed on a mold to provide the sheet with the desiredstructure 407. During molding, the sheet may be inverted so that thebasement membrane of the arterial section constitutes the outer surfaceof the device. In some embodiments, however, the basement membrane maynot constitute the outer surface of the device until it is wrappedaround the connective tissue. After the new structure has been achieved,the device is then irradiated to fix the structure 408. In this sampleprotocol, radiation provided during the fixing step also serves tosterilize the device.

In certain embodiments, devices can be prepared using the followingprotocol: Porcine carotid arteries are harvested by dissection. Afterremoving blood clots, arteries are then decellularized at roomtemperature for 24 hours in 1% (w/v) TRITON-X-100™ that was prepared in10 mM HEPES buffer (pH 8.0) containing 10 mM EDTA. Decellularizedarteries are then washed with 0.9% saline to remove the residualdetergent until foam is no longer observed. Arteries are then treatedfor 24 hours in second HEPES buffer solution (10 mM, pH 7.4) containing30 units/mL DNase, 50 μg/ml gentamicin, 2 mM calcium chloride and 2 mMmagnesium chloride. The DNase solution is discarded, and tissue iswashed three times with 0.9% saline (10 min each time). Decellularizedarteries are further treated in phosphate-buffered saline (pH 6.5)containing 0.2 unit/mL α-galactosidase and 50 mM EDTA to eliminateextracellular α-gal epitope. The artery tissue is then cut to desirabledimensions.

It would be understood that variations can be made in the above protocoland still be in accordance with the present invention. In certainembodiments, for example, biological buffers other than HEPES can beused. Suitable biological buffers can include, but are not limited to,PBS, TBS, and MOPS. Furthermore, the amounts of various buffercomponents can be altered ad still be in accordance with the presentinvention. In certain embodiments, the concentration of HEPES buffer (orother buffer such as PBS, TBS, or MOPS) can range from 1 to 100 mM. Incertain embodiments, the amount of TRITON-X-100™ can range from 0.1 to10% (w/v). In certain embodiments, the amount of DNase can range from 1to 100 units/ml, 1 to 50 units/ml, 1 to 40 units/ml, 4 to 40 units/ml,and 10 to 30 units/ml DNase. In certain embodiments, the buffercomprises 1 to 200 ug/ml gentamicin. In certain embodiments, the amountof calcium chloride and magnesium chloride each may range from 0.1 mM to100 mM, 0.1 to 50 mM, 1 to 50 mM, 1 to 30 mM, 1 to 10 mM, and 0.1 to 5mM. In certain embodiments, the amount of α-galactosidase used to removeα-gal epitopes can range from 0.1 to 10 units/ml, 0.1 to 8 units/ml, 0.1to 5 units/ml, 0.1 to 1 unit/ml, and 0.1 to 0.5 units/ml.

The device prepared by the methods described above can be used invarious ways associated with the repair, regrowth, and regeneration ofvarious types of connective tissue, including tendons and ligaments.Methods of treatment using the device include selecting a tendon orligament in need of treatment, selecting an arterial tissue matrix fromwhich substantially all the native cells have been removed, andcontacting the arterial tissue matrix with at least a portion of thetendon or ligament in need of treatment. Connective tissue in need oftreatment, including tendons and ligaments in need of treatment, can bereadily identified by those skilled in the art, but can include strainedor torn tissue, or tissue having a gap, hole, or some other defect.Furthermore, the device can be applied to anywhere tendons and ligamentsare found, including, but not limited to shoulders, ankles, elbows,knees, fingers, wrists, and feet.

In some embodiments, positioning the device across the defective tissuecomprises wrapping the arterial tissue matrix around the area in need oftreatment. Because the arterial section has already been cut into asheet, it can be wrapped around an injured tendon or ligament. Thewrapped sheet forms a protective sheath, tube, or conduit thatfacilitates the healing process. Without being bound to theory, it isbelieved that the conduit provides a barrier that minimizes the loss ofgrowth factors or other natural cytokines released during the healingprocess. Furthermore, the surface of the device comprising the basementmembrane can be positioned on the outside or inside of the device. Aspreviously discussed, the basement membrane is typically found on theinside of arteries, providing a smooth surface that facilitates bloodflow. In certain instances, however, it may be desirable that thebasement membrane is positioned at the outside surface of the device.When placed on the outside, the smooth surface of the basement membranecan be used to prevent adhesion. This may be useful when damage to theconnective tissue is relatively severe and will take longer to heal.Once the device has been positioned as desired, it can be held in placeusing sutures or various biocompatible adhesives, such as fibrin glue.

The placement of the device around a damaged connective tissue is shownin FIG. 1. A human hand 101 has been surgically cut open. The opening inthe hand 102 is held open with surgical implements 103, exposing thetendons 104 in the wrist. One of the tendons 104 is in need oftreatment, as indicated by a tear 105. A sheet of arterial tissue matrix106 prepared by the disclosed methods is wrapped around the tear 105.The arterial tissue matrix 106 comprises an interior surface 107, whichis adjacent to the tendon 104, and an exterior surface 108. Either theinterior surface 107 or exterior surface 108 may comprise the surfacewith the intact basement membrane, according to certain embodiments.Once the arterial tissue matrix 106 has been wrapped around the tendon104, the arterial tissue matrix 106 is sutured in place.

What is claimed is:
 1. A method for treating a tendon or ligament, comprising: selecting a tendon or ligament in need of treatment; selecting an arterial tissue matrix from which substantially all the native cells have been removed, wherein the arterial tissue matrix comprises an intact basement membrane along the luminal side of an artery from which the tissue matrix is produced; and contacting the arterial tissue matrix with at least portion of the tendon or ligament in need of treatment by wrapping the arterial tissue matrix around the tendon or ligament with the basement membrane facing away from the tendon or ligament.
 2. The method of claim 1, wherein contacting the arterial tissue matrix with at least portion of the tendon or ligament in need of treatment comprises wrapping the arterial tissue matrix around the area in need of treatment.
 3. The method of claim 1, wherein the arterial tissue matrix comprises a porcine arterial tissue matrix.
 4. The method of claim 1, wherein the tendon or ligament is located in an area of the body selected from an ankle, elbow, knee, finger, wrist, shoulder, or foot.
 5. The method of claim 1, wherein the tendon or ligament is selected from a rotator cuff tendon, an anterior cruciate ligament, and a flexor or extensor tendon of the hand, wrist, ankle, or foot.
 6. The method of claim 1, wherein the tendon or ligament in need of treatment comprises an injury selected from a strain, a tear, or a gap.
 7. The method of claim 1, further comprising adding a bioactive substance to the tissue matrix.
 8. The method of claim 7, wherein the bioactive substance comprises an antimicrobial agent.
 9. The method of claim 7, wherein the bioactive substance comprises a cytokine.
 10. The method of claim 7, wherein the bioactive substance comprises a growth factor.
 11. The method of claim 7, wherein the bioactive substance comprises adipose tissue.
 12. The method of claim 7, wherein the bioactive substance comprises cells.
 13. The method of claim 12, wherein the cells comprise stem cells.
 14. The method of claim 13, wherein the stem cells comprise mesenchymal stem cells.
 15. The method of claim 1, further comprising adding a bioactive substance to the tissue matrix, the bioactive substance comprising one or more of an anti-microbial agent, cells, adipose tissue, growth factors, anti-inflammatory agents, steroids, and corticosteroids.
 16. The method of claim 1, wherein the matrix is a porcine matrix.
 17. The method of claim 16, wherein the matrix is derived from a pig that has been genetically modified to reduce or eliminate expression of α-1, 3-galactose moieties.
 18. The method of claim 17, wherein the pig lacks expression of α-galactosyltransferase.
 19. The method of claim 1, wherein the arterial tissue matrix includes elastins present in the arterial section from which the tissue matrix is derived. 